This research project focuses on cell cycle checkpoints as major determinants of chemosensitivity. We also aim to utilize emerging knowledge of these checkpoint control systems to design new chemotherapeutic stratagems for cancer treatment. We are elucidating the checkpoints that arrest human cells in G1 and G2 phases following DNA damage, and uncovering defects in these systems in cancer cells. We hope to trace these systems from the point where DNA damage or unreplicated DNA is sensed by the cell to the response elements that arrest the cell cycle, and in some cases induce apoptosis. We are searching for components of these systems that could be assayed to predict checkpoint integrity and chemosensitivity. Our results suggest that p53 gene mutations prevent cells from arresting in G1 following DNA damage and increase the likelihood of resistance to these agents. This resistance is due, at least in part, to an evasion of p53-mediated apoptosis, although other changes also occur in the mutant p53 cells. We are exploring the interaction of the p53 regulated gene product WAF1/CIP1 with the G1/S cyclin dependent kinases to better define the significance of this protein in G1 arrest, DNA repair and apoptosis. We are investigating both the mechanism of G2 arrest and integrity of the G2 checkpoint in cancer cells. This checkpoint appears to protect cells from DNA damage by extending the time available for DNA repair. We have found DNA damage suppresses activation of the cyclin A/Cdc2 and cyclin B/Cdc2 kinases by maintaining inhibitory phosphorylations on Cdc2. Consistent with this observation we have found that activation of the Cdc25C phosphatase is suppressed in cells treated with DNA damaging agents. We are investigating the mechanism of Cdc25C activation to better define the pathway by which unreplicated and damaged DNA arrest cells in G2 phase. We have applied flow cytometric assays to test the integrity of the G1 and G2 checkpoints in cell lines of the NCI- Anticancer Drug Screen and found that major differences exist. We are presently probing for relationships between the integrity of these- checkpoints and chemosensitivity.